Nonsteroidal Anti-inflammatory Drug Etodolac Research Articles

NSAID Sulindac and Its Analog Bind RXRα and Inhibit RXRα-Dependent AKT Signaling

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their anticancer effects through cyclooxygenase-2 (COX-2)-dependent and independent mechanisms. Here, we report that Sulindac, an NSAID, induces apoptosis by binding to retinoid X receptor-alpha (RXRalpha). We identified an N-terminally truncated RXRalpha (tRXRalpha) in several cancer cell lines and primary tumors, which interacted with the p85alpha subunit of phosphatidylinositol-3-OH kinase (PI3K). Tumor necrosis factor-alpha (TNFalpha) promoted tRXRalpha interaction with the p85alpha, activating PI3K/AKT signaling. When combined with TNFalpha, Sulindac inhibited TNFalpha-induced tRXRalpha/p85alpha interaction, leading to activation of the death receptor-mediated apoptotic pathway. We designed and synthesized a Sulindac analog K-80003, which has increased affinity to RXRalpha but lacks COX inhibitory activity. K-80003 displayed enhanced efficacy in inhibiting tRXRalpha-dependent AKT activation and tRXRalpha tumor growth in animals.

SDX-308, a nonsteroidal anti-inflammatory agent, inhibits NF-κB activity, resulting in strong inhibition of osteoclast formation/activity and multiple myeloma cell growth

AbstractMultiple myeloma is characterized by increased osteoclast activity that results in bone destruction and lytic lesions. With the prolonged overall patient survival achieved by new treatment modalities, additional drugs are required to inhibit bone destruction. We focused on a novel and more potent structural analog of the nonsteroidal anti-inflammatory drug etodolac, known as SDX-308, and its effects on osteoclastogenesis and multiple myeloma cells. SDX-101 is another structural analog of etodolac that is already used in clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Compared with SDX-101, a 10-fold lower concentration of SDX-308 induced potent (60%-80%) inhibition of osteoclast formation, and a 10- to 100-fold lower concentration inhibited multiple myeloma cell proliferation. Bone resorption was completely inhibited by SDX-308, as determined in dentin-based bone resorption assays. SDX-308 decreased constitutive and RANKL-stimulated NF-κB activation and osteoclast formation in an osteoclast cellular model, RAW 264.7. SDX-308 effectively suppressed TNF-α–induced IKK-γ and IκB-α phosphorylation and degradation and subsequent NF-κB activation in human multiple myeloma cells. These results indicate that SDX-308 effectively inhibits multiple myeloma cell proliferation and osteoclast activity, potentially by controlling NF-κB activation signaling. We propose that SDX-308 is a promising therapeutic candidate to inhibit multiple myeloma growth and osteoclast activity and that it should receive attention for further study.

R-Etodolac decreases β-catenin levels along with survival and proliferation of hepatoma cells

Inhibition of hepatoma cells by cyclooxygenase (COX)-2-dependent and -independent mechanisms has been shown previously. Here, we examine the effect of Celecoxib, a COX-2-inhibitor and R-Etodolac, an enantiomer of the nonsteroidal anti-inflammatory drug Etodolac, which lacks COX-inhibitory activity, on the Wnt/beta-catenin pathway and human hepatoma cells. Hep3B and HepG2 cell lines were treated with Celecoxib or R-Etodolac, and examined for viability, DNA synthesis, Wnt/beta-catenin pathway components, and downstream target gene expression. Celecoxib at high doses affected beta-catenin protein by inducing its degradation via GSK3beta and APC along with diminished tumor cell proliferation and survival. R-Etodolac at physiological doses caused decrease in total and activated beta-catenin protein secondary to decrease in its gene expression and post-translationally through GSK3beta activation. In addition, increased beta-catenin-E-cadherin was also observed at the membrane. An associated inhibition of beta-catenin-dependent Tcf reporter activity, decreased levels of downstream target gene products glutamine synthetase and cyclin-D1, and decreased proliferation and survival of hepatoma cells was evident. The antitumor effects of Celecoxib (at high concentrations) and R-Etodolac (at physiological doses) on HCC cells were accompanied by the down-regulation of beta-catenin demonstrating a useful therapeutic strategy in hepatocellular cancer.

Cytotoxic Effect of R-Etodolac (SDX-101) in Combination with Purine Analogues or Monoclonal Antibodies on Ex-Vivo B-Cell Chronic Lymphocytic Leukemia Cells.

Background: SDX-101 (R-etodolac) is the R isomer of the non-steroidal anti-inflammatory drug Etodolac. SDX-101 decreased the in vitro survival of B-cell chronic lymphocytic leukemia (B-CLL) cells and showed synergistic activity with chlorambucil. In multiple myeloma cells, SDX-101 showed cytotoxic activity and displayed a synergistic cytotoxic effect with dexamethasone. Currently, SDX-101 is being tested in phase II clinical trial for treatment of refractory B-CLL in combination with chlorambucil.Aims: To evaluate the ex-vivo cytotoxic effect of SDX-101 in combination with agents proven to be effective first line treatment in B-CLL: fludarabine (FA), cladribine (2-chlordeoxyadenosine, 2-CdA), anti-CD-52 (alemtuzumab, ALT) or anti-CD-20 (rituximab, RTX).Methods: Tumor cells were obtained from 20 untreated patients with B-CLL. Cytotoxicity of SDX-101 and other study drugs was assessed using a propidium iodide-based flow cytometry assay. Apoptosis was assessed measuring activity of caspase-3 and DNA-content (sub-G1 fraction) by flow cytometry, and by measuring protein expression of p53 protein family members (p53 and p73), and BCl-2 family members (pro-apoptotic Bax, Bak and Bid; anti-apoptotic Bcl-2 and Mcl-1). The percentage of apoptotic cells and the expression of apoptosis-regulating proteins were measured at 0 h and 24 h. IC50 values were defined as the concentration of agents that achieved 50% decrease in cell viability. The combination index (CI) was used to estimate sub-additive (CI value 1.2), additive (0.8–1.2) or synergistic (<0.8) interactions.Results: Dose-ranging experiments indicated that the SDX-101 IC50 in CLL cells was 800 μM, and that 400 μM of SDX-101 reduced CLL viability by 11% on average after 24 hrs. An SDX-101 dose of 400 μM was selected for combination studies with FA (used at 3.5 μM, yielding a viability reduction of 9.9%), 2-CdA (0.175 μM, 10.1%), RTX (20μg/ml, 6.1%), and ALT (20μg/ml, 8.4%). The combination of SDX-101+FA provided a drop of 20.9% in cell viability (p<0.03, vs. single drugs alone). The SDX-101+2-CdA combination reduced viability by 27.8% (p<0.006 vs. single drugs), SDX-101+RTX reduced viability by 22.3% (p<0.01 vs. single drugs), and that of SDX-101+ALT combination reduced it by 18.7% (n.s.). The CIs for those combinations were 0.89, 1.17, 0.95, and 1.25, respectively. Apoptosis analysis by sub-G1 fraction and caspase-3 activity confirmed the viability results for each tested combination. Analysis of apoptosis-regulating proteins showed, that SDX-101 combined with RTX or 2-CdA distinctly up-regulated Bax (p<0.02, vs. single drugs), whereas other combinations did not significantly increase its expression. The expression of p73 protein showed 2–4 fold increase after treatment with SDX-101 alone, and all combinations further up-regulated p73, especially that with RTX (up to 6-fold increase in p73 expression). Interestingly, although SDX-101 (400 μM at 24 hr) increased protein expression of Mcl-1, SDX-101 combination with RTX, 2-CdA or FA caused a 2-3-fold decrease of its expression.Conclusion: SDX-101 in combination with low doses of either 2-CdA, FA or RTX exerts an additive cytotoxic effect on ex-vivo B-CLL cells, indicating a possible clinical benefit of such a combination treatment. Up-regulation of pro-apoptotic protein expression (p73 and Bax), and down-regulation of anti-apoptotic protein (Mcl-1) seem to play a mechanistic role in these additive cytotoxic effects.

The R -enantiomer of the nonsteroidal antiinflammatory drug etodolac binds retinoid X receptor and induces tumor-selective apoptosis

Prostate cancer is often slowly progressive, and it can be difficult to treat with conventional cytotoxic drugs. Nonsteroidal antiinflammatory drugs inhibit the development of prostate cancer, but the mechanism of chemoprevention is unknown. Here, we show that the R-enantiomer of the nonsteroidal antiinflammatory drug etodolac inhibited tumor development and metastasis in the transgenic mouse adenocarcinoma of the prostate (TRAMP) model, by selective induction of apoptosis in the tumor cells. This proapoptotic effect was associated with loss of the retinoid X receptor (RXRalpha) protein in the adenocarcinoma cells, but not in normal prostatic epithelium. R-etodolac specifically bound recombinant RXRalpha, inhibited RXRalpha transcriptional activity, and induced its degradation by a ubiquitin and proteasome-dependent pathway. The apoptotic effect of R-etodolac could be controlled by manipulating cellular RXRalpha levels. These results document that pharmacologic antagonism of RXRalpha transactivation is achievable and can have profound inhibitory effects in cancer development.

Efficacy of transdermal nitroglycerin combined with etodolac for the treatment of chronic post-thoracotomy pain: An open-label prospective clinical trial

Chronic post-thoracotomy pain (CPP) is associated with surgical intercostal nerve injury. Like other forms of neuropathic pain, there is no ideal treatment. Nitroglycerin (NTG) has been found efficacious in acute pain, but has not been tested for chronic pain with neuropathic characteristics. The present study investigated the efficacy of NTG combined with the nonsteroidal anti-inflammatory drug etodolac for the treatment of CPP. Thirty of 129 patients who underwent thoracotomy within an 18-month period had moderate to severe pain that did not respond to etodolac. NTG, 5 mg/day, was added to the treatment. A significant reduction in VAS was observed on day 14 of treatment (from 66.7±11 to 42.1±5, P<0.05). Similar changes were noted in breakthrough pain intensity and sleep efficiency. The only side effect was mild headache, which was self-limited to the first few days of NTG administration. We conclude that NTG added to etodolac appears to be effective for the treatment of CPP, with minimal side effects. Further randomized blinded studies are required.

Isolation of an unknown metabolite of the non-steroidal anti-inflammatory drug etodolac and its identification as 5-hydroxy etodolac.

The non-steroidal anti-inflammatory drug etodolac is extensively metabolized in the liver. Renal elimination of etodolac mainly as glucuronide and its other phase I and phase II metabolites is the primary route of excretion. High-performance liquid chromatography assays of human urine after application of etodolac indicated the existence of a further monohydroxylated metabolite (metabolite X) that was identified as 5-hydroxy etodolac. For the identification, electrospray ionization mass spectrometry (ESI-MS) as well as 1H-nuclear magnetic resonance (1H-NMR) and 13C-NMR spectroscopy have been used.

Capillary electrochromatography–electrospray ionization mass spectrometry for the qualitative investigation of the drug etodolac and its metabolites in biological samples

On-line coupling of capillary electrochromatography (CEC) with electrospray ionization (ESI) mass spectrometry (MS) was used for the qualitative investigation of the biotransformation of the non-steroidal anti-inflammatory drug etodolac. The coupling of this electro-driven separation technique with mass spectrometry allowed us to demonstrate the presence of different metabolites of etodolac extracted from human urine.

Separation and identification of etodolac and its urinary phase I metabolites using capillary electrochromatography and on-line capillary electrochromatography–electrospray ionisation mass spectrometry coupling

Capillary high-performance liquid chromatography (capillary HPLC), pressure-assisted capillary electrochromatography (pCEC) and capillary electrochromatography (CEC) were performed in the same capillary packed with 5 μm octadecylsilica (C 18) as stationary phase. These three separation modes were compared from the viewpoint of peak efficiency and separation selectivity in order to critically evaluate the advantages which CEC may offer compared to capillary HPLC for the solution of practical biomedical problems. The separation of the non-steroidal anti-inflammatory drug etodolac (ET, 1) and its phase I metabolites, 6-hydroxy etodolac (6-OH-ET, 2), 7-hydroxy etodolac (7-OH-ET, 3) and 8-(1′-hydroxyethyl) etodolac (8-OH-ET, 4) was selected as an example. Baseline separation of all compounds was achieved in different modes and conditions. The effect of pure electrophoretic separation mechanism on the overall separation selectivity observed in CEC has been shown. A high electroosmotic flow (EOF) was observed in C 18 packed capillary even at pH 2.5 in various buffers. Furthermore, these separations were coupled on-line with electrospray ionisation mass spectrometry (ESI-MS) and the parent drug and its metabolites were identified in urine. For the coupling of CEC with ESI-MS a laboratory-made electrophoretic device was used in order to overcome some technical disadvantages of commercial instrumentation.

Modulation of extracellular matrix metabolism in rabbit articular chondrocytes and human rheumatoid synovial cells by the non-steroidal anti-inflammatory drug etodolac. I: Collagen synthesis

Cultures of human rheumatoid synovial cells and rabbit articular chondrocytes were exposed to various concentrations of Etodolac (from 0.01 to 10 micrograms/ml) in presence or absence of 500 pg/ml (5 U/ml) human recombinant Interleukin-1 beta (IL-1 beta). Incubation of chondrocytes with Etodolac for 24 h did not alter collagen biosynthesis. In contrast, 1 micrograms/ml Etodolac caused a 20% increase of collagen production in synoviocytes. Addition of Etodolac in combination with IL-1 could partially suppress the inhibitory effect exerted by the cytokine on both cell types. Four-day exposure of chondrocytes to 0.1 and 1 micrograms/ml Etodolac led to an increased accumulation of collagen in the cell layer compartment. However, this treatment could not prevent the inhibitory effect of IL-1 on this collagen fraction. Treatment of synoviocytes for eight days with the same concentrations of Etodolac did not modify their collagen production but suppressed totally the inhibitory effect of IL-1. These data show that Etodolac is able to augment chondrocyte metabolism during a long term treatment. Moreover, under certain conditions, this drug can reduce or even suppress the IL-1-induced inhibition of collagen biosynthesis, a process that may take a part in the connective tissue alterations associated with osteoarticular diseases such as rheumatoid arthritis and osteoarthritis.

Modulation of extracellular matrix metabolism in rabbit articular chondrocytes and human rheumatoid synovial cells by the non-steroidal anti-inflammatory drug etodolac. II: Glycosaminoglycan synthesis

The effect of Etodolac on glycosaminoglycan (GAG) synthesis by human synovial cells and rabbit articular chondrocytes in culture was studied at doses ranging from 0.01 to 10 micrograms/ml. In chondrocyte cultures, short-term exposure to Etodolac decreased the total amount of GAGs (mainly chondroitin sulfate) affecting essentially the cell layer-associated fraction. When cells were incubated with Interleukin-1 (IL-1), Etodolac still exerted its inhibiting effect on GAG synthesis. Long-term exposure of chondrocytes to Etodolac caused a diminution of the total GAG synthesis for the highest concentration studied (1 micrograms/ml). IL-1 alone or in combination with Etodolac decreased the synthesis of GAGs suggesting that pretreatment with Etodolac cannot prevent the action of IL-1. In synoviocyte cultures, the drug caused an inhibition of the GAG biosynthesis (mainly hyaluronic acid). In presence of IL-1, Etodolac at the highest concentration partially diminished the stimulatory effect of IL-1. A 8-day exposure of the cells to Etodolac led to a decrease of GAG synthesis. In the same experiment, IL-1 alone caused a slight increase of GAG production that was not abolished by Etodolac treatment.